A Primer on LC/NMR/MS

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  • Published: Feb 15, 2014
  • Channels: HPLC / Base Peak / NMR Knowledge Base
thumbnail image: A Primer on LC/NMR/MS

In this primer, Steve Down provides a detailed look at how these analytical techniques have been coupled together to provide an extremely powerful bioanalytical tool.


Recently, the world's first fully integrated LC/NMR/MS system was launched. Hailed as the 'ultimate' bioanalysis tool for the pharmaceutical and biotechnology industries, this new technology takes advantage of mass spectrometry's rapid and ultra-sensitive screening capabilities which can identify peaks of interest in complex mixtures for further analysis by NMR. The ability of knowing ahead of time which peak to submit to extended 1D or 2D NMR analysis, greatly improves the efficiency and information content from a chromatographic separation.


Table of Contents

1. Introduction
2. LC/NMR - The Basics
3. LC/MS - The Basics
4. Linking LC/NMR and LC/MS
5. Applications of LC/NMR/MS
6. Future Developments
7. Abbreviations
8. References
9. Acknowledgements


1. Introduction

In the field of analytical chemistry, there are many well-established techniques for identifying unknown compounds. Many of these are single techniques such as nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy, in which the sample is analyzed directly. However, for mixtures, initial separation of the components before analysis greatly facilitates detection and identification by separating overlapping components. This can be achieved with simple systems such as gas chromatography (GC) combined with flame ionization, (FID), flame photometric (FPD) and nitrogen/phosphorus (NPD) detectors, as well as the so-called hyphenated systems, in which spectroscopic detectors are used. These include high-performance liquid chromatography (HPLC) combined with ultraviolet (UV), fluorescence, NMR and mass spectrometry (MS) detectors.

Combined liquid chromatography/mass spectrometry (LC/MS) has advanced rapidly from its early development in the 1970s and is now a standard technique (References 1-5). There are many commercial systems available and the detection sensitivity can reach very low levels. For drug detection and metabolism and environmental studies, the commonly required detection limits in the ng-m g range are readily attainable.

Combined liquid chromatography/nuclear magnetic resonance spectroscopy (LC/NMR) was proposed over 20 years ago, but did not capture the imagination of researchers/developers due to its inherently poor sensitivity. Relatively recent design improvements have improved sensitivity and the technique is now established (References 6,7).

In practice, use of either LC/MS or LC/NMR alone can often solve analytical problems but there are occasions when they fail and extra techniques are required to solve the problem. In these cases, using both LC/MS and LC/NMR can provide extra information and structural identity. It seems a natural progression to combine the two systems into one integrated LC/NMR/MS system.


2. LC/NMR - The Basics

LC/NMR was first proposed in the late 1970s but took off only when instrumental developments such as high magnetic field, better coil design and solvent suppression techniques permitted detection limits to be lowered to useful levels.

Typical layout of a LC-NMR system.


The eluent from the HPLC column generally passes through a flow cell for UV detection, in order to determine when the analyte peaks are eluting. The flow passes to a probe that sits within the NMR magnet. There are several types of probe design but the key in all cases is to wind the coil directly around the cell itself, to reduce the gap between the coil and the cell. This maximises the ratio between the volume of the cell and the volume of the coil (known as the filling factor) and increases the signal-to-noise ratio.

There are three main modes of operation in the LC-NMR system: continuous-flow, stopped-flow and loop collection. In continuous-flow mode, all the column eluent flows to the cell continuously during the HPLC run, and NMR spectra are recorded over the whole time. One advantage of this approach is that all components are sampled, including those that do not give UV signals. However, relatively high concentrations of analyte are required (1-5 m g on column) depending on field strength.

In stopped-flow mode, the HPLC flow is stopped while NMR spectra are recorded for a particular HPLC peak, then is turned on again. A series of peaks may be measured this way during the run. The UV detector, or prior knowledge of HPLC retention times, is generally used to signal when to stop the flow. In this way, longer NMR acquisition times, traditional NMR techniques (such as two-dimensional NMR) and smaller component concentrations can be accommodated. Stopping the flow does not usually affect chromatographic resolution.

For loop collection, the HPLC fractions are collected in their own capillary loops during the HPLC run. After the run is completed, the individual loops are eluted for NMR analysis. This is useful when long analysis times are required, for example with small amounts of sample (less than 500 ng) or in 2-dimensional experiments. The loops can also be removed and stored for later or repeated analysis. Alternatively, the fractions can be collected from a different HPLC instrument and brought to the LC-NMR instrument for analysis.

The advantages of LC/NMR include the ability to distinguish between isomers, whether structural, conformational or optical. Also, the method is non-destructive, so fractions can be recovered after analysis and stored, if required, for later experiments. The volatility of the buffers is not an issue. Unlike in LC/MS, both volatile and non-volatile buffer systems are suitable.


3. LC/MS - The Basics

LC/MS development began in earnest in the 1970s, when these 2 seemingly incompatible techniques, linking separation in a liquid at atmospheric pressure to detection in a vacuum, were combined. The early interfaces for accomplishing this, such as direct liquid introduction, or a moving belt, led the way for the common interfaces used today that seem so indispensable to many labs: electrospray, atmospheric pressure chemical ionization (APCI), particle beam (PB) and thermospray.

Today, the most popular interfaces are electrospray and APCI, contributing around 74% and 12%, respectively, of the published literature involving LC/MS and liquid flow soft ionization techniques (Reference 13). In electrospray, the analyte in solution is sprayed at atmospheric pressure from a hollow needle that is charged at its tip to typically 2-5 kV. This produces a spray of charged droplets, which are evaporated to induce ion formation. So ionization occurs in the liquid phase.

In APCI, the solution is again sprayed from a hollow needle, but the spray enters a heated chamber (typically at 400°C) and a corona discharge in the gas produces charged clusters of water. These react with the analyte molecules (chemical ionization) to produce analyte ions. Ionization occurs in the gas phase.

There are several types of ion detector employed in the mass spectrometer. These include the single quadrupole, triple quadrupole, ion trap, Fourier transform (FT) ICR and time-of flight (TOF) detectors, as well as various hybrids.

Many applications require only the simplest of information from the MS experiment, namely the molecular mass of the analyte. This can be measured with the ion trap and single quadrupole instruments, which are also the least expensive on the market. These instruments can also be used to obtain fragmentation patterns, by increasing the so-called cone voltage (nozzle-skimmer or orifice-skimmer difference) in the quadrupole or by multi-stage MS in the ion trap. Although they are generally operated at unit mass resolution, single quadrupoles can give detailed structural information in many cases.

For more specific experiments, ion traps and the multi-stage instruments such as the triple quadrupole mass spectrometers are used. These permit a more detailed examination of the fragmentation mechanism than single quadrupoles by using techniques like precursor-ion and product-ion scanning, selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) in which particular steps in the fragmentation are monitored. When accurate masses are required, for example to determine the elemental compositions of molecular ions and fragments, and to distinguish between isobaric ions, high-resolution instruments such as the quadrupole-TOF and FT ICR mass spectrometers can be used.


4. Linking LC/NMR and LC/MS

There are effectively two ways of linking HPLC to two detector systems: in series and in parallel. With the former combination, the effluent from the column goes to each detector in turn. This has the disadvantage that is it much more difficult to correlate the results from each system for a particular component in a mixture, because it is analyzed at different times in each detector. Additionally, series connection can induce leaks from the NMR flow probe caused by pressure differences between the NMR and MS systems.

With a parallel system, the flow from the HPLC is split between both detectors. This makes it easy to adjust the split flows, depending on the type of experiment required. For example, an analyte can be detected simultaneously in both systems. Alternatively, the MS data, which can be acquired rapidly, may be used to direct the NMR experiments, for example to concentrate on one particular eluting peak.

In series and parallel systems, it is relatively easy to disconnect the mass spectrometer from the NMR spectrometer, if the former is required for separate experiments. Since a dedicated LC/NMR/MS instrument may be considered a luxury in some labs, disconnection may be a prerequisite.

It is also advantageous to deploy a UV detector after the HPLC column. If the UV cell is positioned before the splitter, it can be used to initiate the next step when an analyte is detected: begin NMR or MS detection, or synchronise delivery of the peak to the MS and NMR detectors.

Typical layout of a LC-NMR-MS system.


If the UV detector is positioned after the splitter, the eluent can be directed to the mass spectrometer and a peak observed before that peak reaches the UV cell. When that same peak is subsequently detected at the cell, it can be used to initiate a stopped-flow experiment on the peak in the NMR instrument.

Bearing in mind that they are two very different detection techniques, linking the LC-MS and LC-NMR systems is relatively simple, but there are other practical problems to overcome: solvent compatibility, instrumental sensitivity and magnetic field effects.

In LC/NMR, one of the main concerns is how to suppress the NMR signals from the solvent, which would otherwise mask the analyte signals. This can be achieved using solvents that minimise contributions to the 1H NMR spectrum, such as acetonitrile in D2O that is pH-adjusted with sodium phosphate. This is fine for LC/NMR alone but causes problems in MS due to deposition of the phosphate salts in the source. Alternative modifiers for the solvent include aqueous trifluoroacetic acid, but this again creates problems in the mass spectrometer by suppressing ionization of acidic analytes. Formic acid (pH-adjusted with ammonium formate) is used in many experiments because it gives a single proton resonance in NMR away from most analytes (around 9 ppm) and does not suppress ionization of acids in the mass spectrometer.

To eliminate the proton NMR signals altogether, deuterated solvents, such as D2O, acetonitrile-d3 and methanol-d4 are generally used. However, they cause problems by initiating H/D exchange with the analyte, so giving a false measurement of molecular mass by MS. On the other hand, H/D exchange can be exploited in MS to determine the number of exchangeable H atoms in the analyte. If the occurrence of H/D exchange is considered undesirable in a particular experiment, it can be reversed by the post-column addition of protic solvents (H2O, MeOH) to the flow to initiate back exchange.

In order to remove any interferences from solvent signals during NMR analysis, solvent suppression techniques are employed, the main ones being presaturation and WET (Water suppression Enhanced through T1 effects). The former is a long-standing method that uses shaped pulses to saturate the solvent resonance(s). The WET method uses selective pulses to excite the solvent resonances then dephasing gradient pulses to destroy them. The two techniques take 0.5-2 s and 50-100 ms, respectively, so the WET method is preferred for continuous-flow NMR.

One major disadvantage with NMR is its insensitivity: relatively large amounts of analyte are required to give good signals, compared with MS. For proton NMR, 1-5 micro-g on-column in flow mode will produce a good signal, whereas only ng amounts or less are needed for MS. This difference is overcome by splitting the column flow after the UV detector, with 2-5% being directed to the mass spectrometer and the remainder to the NMR spectrometer.

NMR instruments operate with a strong magnetic field that can interfere with the mass spectrometer operation (typically 400-600 MHz, but up to 800 MHz). There is much anecdotal evidence of unusual MS results that could not be explained until it was realised that an NMR machine was in the proximity (e.g. behind a wall in the next lab). Modern superconducting NMR magnets are actively shielded, so this problem should not occur but for lab-built systems using older NMR instruments, the positioning of the mass spectrometer away from the NMR magnet is crucial.

For optimum sensitivity in NMR, good HPLC separation with sharp eluting peaks is essential. This will also help to avoid co-interference between the eluting components, although signals from simple mixtures of a few components can be resolved. The level of separation employed is also good enough for mass spectrometry. In many experiments, the mass spectrometer is used to determine molecular masses only, in order to confirm the structure determined by NMR spectroscopy. In these cases, ion trap or quadrupole mass spectrometers can be employed. These are also the most common instruments on the market that many labs have, so the main components for a combined system may be already in place.

Since NMR or MS alone solves about 80-90% of the analytical problems, the combined LC-NMR-MS system need only be connected up for those 10-20% of insoluble problems. For labs with limited budgets, this is the preferred solution since the instruments are not tied up. For those organisations with a bigger spend, one system can be dedicated to LC/NMR/MS.

Compared with a commercial system, with which extensive training is usually given by the manufacturer as part of the deal, in-house systems come with no such training. This can be one of the major obstacles. Most technicians are specialists in NMR spectroscopy or mass spectrometry, but are not proficient in both techniques. This can create difficulties for operating the combined system, as well as in interpreting the data. Therefore a system that runs under full automation is crucial to guarantee success

Currently the only commercial LC-NMR-MS systems are produced by Bruker Analytik and they have sold 24 systems to date over a period of about 18 months. There are different versions with different strength magnets, from 500-800 MHz, but they all currently use the same mass spectrometer, the Esquire ion trap.

A: The HPLC pump, B: The NMR-MS interface, C: The ion trap
mass spectrometer, D: the LC-NMR rack with the DAD/UV detector.


The NMR-MS interface is designed to allow different modes of operation, including separate analysis by either MS or NMR (on-flow or stopped flow) and combined analysis by MS and NMR. The flow to the mass spectrometer can be stopped when large concentrations of a particular component are eluted, to prevent overloading of the mass spectrometer source. There is also a removable loop cartridge containing 36 sample loops that can be used to collect eluting peaks from a separate HPLC system, possibly in another lab, then transport them to the LC-NMR-MS system for elution and analysis.


5. Applications of LC/NMR/MS

Papers from two different research groups were published in close succession to signal the successful application of LC-NMR-MS. In late 1995, an artificial mixture containing the drug fluconazole and two related triazoles was analyzed using a 500-MHz NMR instrument and a quadrupole mass spectrometer (Reference 14). Using isocratic acetonitrile-D2O as mobile phase, the flow was split with 60% directed to the mass spectrometer, operated in particle beam mode.

In early 1996, HPLC-NMR-MS with a 500-MHz NMR machine and an ion trap was used to analyze a real sample: human urine from a subject given acetaminophen. (Reference 15). Eluting with a reversed-phase (RP) gradient from 0.1% trifluoroacetic acid in D2O to 50% acetonitrile-d3 in D2O, the flow was split with 95% going to the NMR machine. MS was conducted in electrospray mode. Glucuronide and sulphate metabolites of the parent drug were identified, as well as the endogenous metabolite phenylacetylglutamine.

Both of these applications were drug-related and most of the remaining published applications are also studies of drugs and their metabolites. However, LC-NMR-MS has been successfully utilised in several other areas, such as the analysis of natural product extracts (flora and fauna), combinatorial peptides mixtures, food extracts and polymers.

Drugs and Metabolites

The metabolism of paracetamol in humans was carried out by the analysis of urine by LC-NMR-MS (Reference 16) using a single quadrupole mass spectrometer. Analyte separation was effected with a gradient of non-deuterated acetonitrile in D2O containing deuterated trifluoroacetic acid and the eluent was split with 2% going to the mass spectrometer. In order to reverse H/D exchange in the analytes, a make-up flow of methanol containing 1% acetic acid was added before MS.

Two studies of the dihydroquinoxalinecarboxylate GW420867, under development for treating HIV infections, have been carried out. In the earlier work (Reference 17), human and animal urines were analyzed after oral dosing. Using RP HPLC with a gradient of acetonitrile in D2O contg. acetic acid, fractions identified by UV spectroscopy were stored in a sampling unit. After NMR analysis on a 600-MHz instrument, the fractions were analyzed by MS in an ion trap, both with and without H/D back exchange. CAD of the protonated (deuterated) parent ions was also carried out.

In the later work (Reference 18), the triazolopyridine GI265080, a potential drug for treating bipolar disorders, was also analyzed in rat urine. Using an ion trap, the flow was analyzed by MS first, to determine when drug-related peaks were eluting and to determine the component molecular masses. This data was used to initiate trapping of the eluting peaks for stopped-flow NMR. Data-dependent MS/MS was also used to gain further structural information and to trigger peak trapping for NMR.

A series of studies on the metabolism of halo-substituted (trifluoromethyl)anilines in the rat by LC-NMR-MS have been reported. For 2-bromo-4-(trifluoromethyl)aniline, again using a gradient of acetonitrile in D2O contg. acetic acid (Reference 19), an ion trap and electrospray MS/MS in the positive mode were employed. The major urinary metabolite was identified as the sulphate conjugate. The value of the combined approach was illustrated here because the sulphate group was silent in NMR. Both 1H and 19F NMR were employed to aid metabolite characterisation. In a later study on the same compound, (Reference 20) using negative-ion electrospray ionization with a single quadrupole, all metabolites containing Br and F atoms were characterised.

The 2-chloro- analogue was also studied by LC-NMR-MS (Reference 21), again using negative-ion electrospray on a single quadrupole and 19F NMR spectroscopy, with a 5:95 split to mass and NMR spectrometers. The sulphate conjugate was confirmed as the major urinary metabolite.

The metabolism of ibuprofen in humans by LC-NMR-MS (Reference 22) used negative-ion MS on a single quadrupole with a 500-MHz NMR machine to identify the hydroxy- and carboxy- metabolites as well their glucuronide conjugates in urine. The glucuronide diastereomers were distinguishable by NMR. A probable artefact resulting from dehydration of a side-chain hydroxylated glucuronide was also identified.

The human metabolism of the HIV-1 reverse transcriptase inhibitor BW935U83, a fluoropentofuranosyluracil derivative, has been studied by a combination of continuous-flow and stopped-flow 1H and 19F NMR spectroscopy and by LC-NMR-MS (Reference 23). The primary urinary metabolite was identified by NMR alone as the glucuronide of the parent drug. An early eluting minor metabolite could not be identified by NMR but was characterised as 3-fluororibolactone by continuous-flow LC-NMR-MS and MS/MS in an ion trap, with simultaneous analysis in both detectors.

The LC-NMR-MS system was extended for the anal. of a synthetic mixture of nonsteroidal antiinflammatory drugs (NSAIDS) by RP-HPLC with on-line UV diode array, FT IR, 1H NMR and TOFMS detection (Reference 24). The system was developed in flow injection mode using model drugs as test analytes (Reference 25). The flow passed in turn through the UV/DAD, an FT IR cell then a UV detector and was split 95:5 to the NMR spectrometer (500 MHz) and a TOF mass spectrometer. The second UV detector was used to evaluate band broadening and to mark passage of the peaks. The TOF mass spectrometer in electrospray ionization mode gave accurate molecular masses leading to elemental compositions. On-flow and stopped-flow NMR spectra were measured. For the model compounds, the detection limits were 50 micro-g. The NSAIDS were eluted with 50% acetonitrile in D2O containing 1% deuterated formic acid and interpretable spectra were obtained from each detector, despite not being fully optimised.

The suitability of superheated heavy water as an eluent for RP-HPLC has been established recently (Reference 26) and its use has been extended to LC-NMR-MS. In one study using a mixture of model drugs (Reference 27), pure superheated D2O proved to be a good eluent. In a second study (Reference 28) phosphate-buffered D2O (pD 3.0) was used to elute a mixture of four sulphonamide drugs with a slow temperature gradient from 160-200°C. Good separation was achieved over 20 min and the NMR spectra were obtained under stopped-flow conditions. The electrospray mass spectra were recorded with a triple quadrupole, but in scan mode only. The spectra showed that the methyl substituents on the pyrimidine ring were susceptible to H/D exchange. The main advantage of this eluent is that is produces no proton NMR signals.

A recent report (Reference 29) described the use of heated and superheated D2O as an eluent in an RP-HPLC-UV(DAD)-IR-NMR-MS system for the analysis of model drugs. The eluent was split, with 95% being directed, in turn to the IR, UV and NMR detectors, using on-flow detection for NMR. The remaining flow was analyzed by APCI MS in a single quadrupole mass spectrometer, with a make-up flow of 90% aqueous methanol to maintain chromatographic resolution and ensure efficient ionization. All types of spectra were readily recorded for analyte levels of 46-500 micro-g on-column.

In another variation of the technique, radio-HPLC-NMR-MS was used to study the metabolism of the drug practolol in the rat, after oral dosing with a mixt. of 14C- and 13C-labelled drug (Reference 30). Separation was accomplished with a gradient of methanol-d4 in D2O contg. 10 mM ammonium formate and the eluent was monitored by UV then radioactivity detection (in-line) before the usual 5:95 split. The radiochromatogram showed which peaks in the UV and mass chromatograms contained metabolites, enabling stopped-flow proton NMR spectroscopy (500 MHz) to be triggered. In positive-ion electrospray MS (triple quadrupole), the radiolabelled peaks were readily detected. A futile deacylation (N-deacetylation/reacetylation) was also observed.


Natural Products

LC-NMR-MS has been used in several studies to identify natural products in plant extracts, which generally consist of complex mixtures. In the first of these (References 31), on-flow proton NMR and electrospray ionization MS in an ion trap were used to characterise ecdysteroids in extracts of Silene otites. After RP-HPLC using D2O in acetonitrile-d3 and UV detection, the flow was split 95:5 for simultaneous detection by NMR and MS. The peaks of interest were analyzed further by stopped-flow NMR to give better quality spectra and confirm structural assignments. In addition, a partially resolved peak was identified.

Several types of compound, including flavonoid glycosides and naphthodianthrones, were identified in an extract of Hypericum perforatum. (Reference 32) All known major constituents were found as well as two novel ones: the arabinoside and galacturonide of quercetin. A typical gradient involving acetonitrile-acetic acid-D2O-ammonium acetate was used, with the standard 95:5 split to a 500-MHz NMR machine and a triple quadrupole mass spectrometer operated in negative-ion electrospray mode.

Flavonoid glycosides in apple peel were identified by LC-NMR-MS (Reference 33), in which either UV detection or MS/MS in an ion trap were used to trigger NMR measurements in flow mode or trapping in the storage unit. Both UV spectroscopy and MS/MS could distinguish between quercetin glycosides and phloretin glycosides. In NMR, the 1D total correlation spectroscopy (TOCSY) method for assignment and structural elucidation in crowded regions of the spectra was employed. Both the flavonoid and glycoside moieties were readily identified from the combined use of NMR and MS data.

A second study on ecdysteroids, this time in an extract of Lychnis flos-coculi, was carried out using RP-HPLC-UV-IR-NMR-MS (Reference 34). The system was developed and optimised as described above (Reference 25). The ecdysteroid components in the extract were identified despite incomplete chromatographic resolution.

The technique has not been limited to natural products in plants. Two related studies examined the toxic steroid glycoside constituents of the starfish Asterias rubens, known as asterosaponins (References 35, 36). The classical extraction and separation techniques used in the earlier method were replaced by a matrix solid-phase dispersion in the later study in order to speed up the process. LC-NMR-MS with on-flow and stooped-flow modes was used. Back-exchange experiments to reverse the H/D exchange occurring during chromatography proved useful. Compounds of molecular mass in the range 1200-1400 Da were identified.


Plant Studies

Two studies on the metabolism of xenobiotic compounds in maize plants have been conducted by HPLC-NMR-MS. In the first (Reference 37), 5-nitropyridone was applied to hydroponically grown plants and the shoots and roots analyzed. The split eluent reached the mass spectrometer before the NMR spectrometer and MS/MS in an ion trap was used to detect the 5-nitropyridone fragment and initiate stopped-flow proton NMR experiments. In this way, the N-glucoside and the N- and O-malonylglucosides of the parent compound were identified.

The second study (Reference 38) involved 5-(trifluoromethyl)pyridone, a model compound for herbicides, in hydroponically grown maize. On-flow 19F NMR spectroscopy and electrospray MS and MS/MS in an ion trap were correlated with stopped-flow proton NMR to identify the peaks in the complicated chromatogram obtained after minimal sample clean up. The two major metabolites were identified as the N-glucoside and O-malonylglucoside.

Miscellaneous

LC-NMR-MS has been tested in different applications, including the analysis of combinatorial libraries (Reference 39) and polymer additives (Reference 40). An early assessment of the technique was carried out on a 9-component peptide library with a RP gradient of acetonitrile in trifluoroacetic acid-D2O. The 5:95 split to the 500-MHz NMR spectrometer and single quadrupole mass spectrometer for on-flow analysis was set up, with MS in electrospray mode. Despite two coeluting components, all peptides were identified.

A mixture of the polymer additives 2,6-di-tert-butyl-4-methylphenol, Irganox 1076 and diisooctyl phthalate was analyzed by LC-NMR-MS and off-line FT IR spectroscopy. Size exclusion chromatography was used, with a mobile phase of chloroform-d containing ammonium acetate-D2O and methanol-d4 to promote ionization. After the 95:5 split, the flow to the NMR instrument was split 50:50, one portion going to an interface to be collected for IR studies.


6. Future Developments

Now that the principle of linking HPLC to more than one detector has been established as a practical reality, there are many possibilities for adding extra detectors to the LC-NMR-MS system. As described in this article, HPLC-UV-NMR-IR-MS has been successfully attempted.

It has been illustrated recently that LC/MS can be linked simultaneously to two MS detectors (ICPMS and TOFMS) for the analysis of drug metabolites in rat urine (References 41, 42). Following on from that, research groups in the UK at AstraZeneca, Micromass and Imperial College of Science, Technology and Medicine are working jointly on running LC-NMR-MS using ICPMS alone and with the dual detectors ICPMS/TOFMS. ICPMS is useful because it can be used to detect specific atoms, such as bromine, arsenic and selenium.

It is also feasible to add other spectroscopic detectors like circular dichroism and fluorescence detectors. It would also be interesting to perform different separation technologies linked to multiple detectors, such as supercritical fluid chromatography (SFC), capillary electrophoresis or capillary electrochromatography. According to Manfred Spraul at Bruker Analytik, their next major development will be the introduction of an HPLC-SPE-NMR-MS system. Solid-phase extraction (SPE) is normally used to preclean samples (either on-line or off-line) before HPLC. In this case, SPE is being used after the HPLC column and peak detector to trap eluting peaks, in a similar way to loop collection. Non-deuterated solvents can be used for trapping, so that the mass spectrum is not affected by molecular mass changes. For NMR, the trap can be flushed with D2O, blown dry and eluted with pure deuterated solvents, to minimise interfering solvent signals.


7. Abbreviations

APCI atmospheric pressure chemical ionization
DAD diode array detector
FT Fourier transform
GC gas chromatography
HPLC high-performance liquid chromatography
ICP inductively coupled plasma
IR infrared
LC/MS liquid chromatography/mass spectrometry
LC-NMR liquid chromatograph-nuclear magnetic resonance spectrometer
LC/NMR liquid chromatography/nuclear magnetic resonance spectrometry
LC-NMR-MS liquid chromatograph-nuclear magnetic resonance spectrometer-mass spectrometer
LC/NMR/MS liquid chromatography/nuclear magnetic resonance spectrometry/mass spectrometry
MRM multiple reaction monitoring
MS mass spectrometry
PB particle beam
NMR nuclear magnetic resonance
RP reversed-phase
SFC supercritical fluid chromatography
SPE solid-phase extraction
SRM selected reaction monitoring
TOF time of flight
UV ultraviolet
WET water suppression enhanced through T1 effects


8. References

For further reading see: On-line LC-NMR and Related Techniques by Klaus Albert.

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2. Cole, R.B. (Editor) Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications, John Wiley & Sons, 1997.

3. Niessen, W.M.A. Liquid Chromatography-Mass Spectrometry. 2nd Edition, Revised and Expanded, Marcel Dekker, 1998.

4. Meyers, R.A. (Editor) Encyclopedia of Analytical Chemistry, John Wiley & Sons, 2000.

5. Lindon, J.C., Tranter, G.E., Holmes, J.L. (Editors) Encyclopedia of Spectroscopy and Spectrometry, Academic Press, 2000.

6. Albert, K. Liquid chromatography-nuclear magnetic resonance spectroscopy. J. Chromatogr. A, 1999, 856, 199-211.

7. Gfrorer, P., Schewitz, J., Pusecker, K., Bayer, E. On-line coupling of capillary separation techniques with 1H NMR, Anal. Chem. 1999, 71, 315A-321A.

8. Taylor, S.D., Wright, B., Clayton, E., Wilson, I.D. Practical aspects of the use of high performance liquid chromatography combined with simultaneous nuclear magnetic resonance and mass spectrometry. Rapid Commun. Mass Spectrom. 1998, 12, 1732-1736.

9. Peng, S.X. Hyphenated HPLC-NMR and its application in drug discovery. Biomed. Chromatogr. 2000, 14, 430-441.

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13. Down, S., Halket, J.M. (Editors) LC/MS: The Applications Database, HD Science Ltd. 2001.

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15. Shockcor, J.P., Unger, S.E., Wilson, I.D., Foxall, P.J.D., Nicholson, J.K., Lindon, J.C. Combined HPLC, NMR spectroscopy and ion trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine. Anal. Chem. 1996, 68, 4431-4435.

16. Burton, K.I., Everett, J.R., Newman, M.J., Pullen, F.S., Richards, D.S., Swanson, A.G. On-line liquid chromatography coupled with high-field NMR and mass spectrometry (LC-NMR-MS): a new technique for drug metabolite structure elucidation. J. Pharm. Biomed. Anal. 1997, 15, 1903-1912.

17. Dear, G.J., Ayrton, J., Plumb, R., Sweatman, B.C., Ismail, I.M., Fraser, I.J., Mutch, P.J. A rapid and efficient approach to metabolite identification using nuclear magnetic resonance spectroscopy, liquid chromatography/mass spectrometry and liquid chromatography/nuclear magnetic resonance spectroscopy/sequential mass spectrometry. Rapid Commun. Mass Spectrom. 1998, 12, 2023-2030.

18. Dear, G.J., Plumb, R.S., Sweatman, B.C., Ayrton, J., Lindon, J.C., Nicholson, J.K., Ismail, I.M. Mass directed peak selection, an efficient method of drug metabolite identification using directly coupled liquid chromatography-mass spectrometry-nuclear magnetic resonance spectroscopy. J. Chromatogr. B 2000, 748, 281-293.

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9. Acknowledgements

The author thanks the following people for their helpful comments and discussions throughout the writing of this article: Ian Wilson of AstraZeneca Pharmaceuticals, Macclesfield, UK; Jeremy Nicholson of Imperial College of Science, Technology and Medicine, London UK; Manfred Spraul of Bruker Analytik GmbH, Silberstreifen (Rheinstetten), Germany.

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